Evolution of Microtubule Organizing Centers Across the Tree of Eukaryotes

Home , Andalucia (Excavata), Breviata, Collodictyonidae, Palpitomonas bilix, Postgaardi, Rictus (bicosoecid)

The Plant Journal (2013) 75, 230–244 doi: 10.1111/tpj.12145

A GLORIOUS HALF-CENTURY OF MICROTUBULES Evolution of microtubule organizing centers across the tree of eukaryotes

Naoji Yubuki* and Brian S. Leander The Departments of Botany and Zoology, Beaty Biodiversity Research Centre and Museum, University of British Columbia, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada

Received 16 December 2012; revised 04 February 2013; accepted 5 February 2013; published online 8 February 2013. *For correspondence (e-mail [email protected]).

SUMMARY

The architecture of eukaryotic cells is underpinned by complex arrrays of microtubules that stem from an organizing center, referred to as the MTOC. With few exceptions, MTOCs consist of two basal bodies that anchor flagellar axonemes and different configurations of microtubular roots. Variations in the structure of this cytoskeletal system, also referred to as the ‘flagellar apparatus’, reflect phylogenetic relationships and provide compelling evidence for inferring the overall tree of eukaryotes. However, reconstructions and subsequent comparisons of the flagellar apparatus are challenging, because these studies require sophisticated microscopy, spatial reasoning and detailed terminology. In an attempt to understand the unifying features of MTOCs and broad patterns of cytoskeletal homology across the tree of eukaryotes, we present a comprehensive overview of the eukaryotic flagellar apparatus within a modern molecular phylogenetic context. Specifically, we used the known cytoskeletal diversity within major groups of eukaryotes to infer the unifying features (ancestral states) for the flagellar apparatus in the Plantae, Opisthokonta, Amoebozoa, Stramenopiles, Alveolata, Rhizaria, Excavata, Cryptophyta, Haptophyta, Apusozoa, Breviata and Collodictyo- nidae. We then mapped these data onto the tree of eukaryotes in order to trace broad patterns of trait changes during the evolutionary history of the flagellar apparatus. This synthesis suggests that: (i) the most recent ancestor of all eukaryotes already had a complex flagellar apparatus, (ii) homologous traits associated with the flagellar apparatus have a punctate distribution across the tree of eukaryotes, and (iii) streamlining (trait losses) of the ancestral flagellar apparatus occurred several times independently in eukaryotes.

Keywords: biodiversity, cytoskeleton, evolution, excavates, ﬂagellar apparatus, protists.

Apusozoa, Breviata, Collodictyonidae, Cryptophyta and A BRIEF OVERVIEW OF EUKARYOTIC DIVERSITY Haptophyta) and ﬁve major clades or ‘supergroups’ [the Eukaryotes that are not plants, animals or fungi, the so- Plantae, Opisthokonta, Amoebozoa, SAR (Stramenopiles, called ‘protists’, are much more diverse than is usually Alveolata and Rhizaria) and Excavata] (Kim et al., 2006; appreciated, particularly in regard to total species numbers Burki et al., 2009; Roger and Simpson, 2009; Ishida et al., and the ultrastructural and genomic complexity of their 2010; Parfrey et al., 2010; Heiss et al., 2011; Adl et al., 2012; cells (Ishida et al., 2010; Parfrey et al., 2010; Adl et al., Zhao et al., 2012). 2012). A comprehensive tree of eukaryotes with resolved Land plants, animals and fungi are each most closely phylogenetic relationships among dozens of very different related to different lineages of single-celled eukaryotes, lineages is beginning to emerge through the rapid accumu- and are nested within different supergroups. For instance, lation and comparative analysis of genomic data (e.g., land plants are nested within the Plantae, which also Parfrey et al., 2010; Burki et al., 2012; Brown et al.,2012).This includes red algae, green algae and glaucophytes (Graham phylogenetic context provides the foundation for inferences and Wilcox, 2000; Archibald and Keeling, 2004) (Figures about the evolution of the eukaryotic cytoskeleton. The 1a–c). Members of this supergroup possess plastids that most widely accepted tree of eukaryotes currently consists were derived directly from a cyanobacterium via (primary) of several minor groups, without a clear sister lineage (the endosymbiosis; glaucophytes still possess a cyanobacterial

Figure 1. Images representing major lineages of eukaryotes. Some photos are modified with (a) (b) (c) permission from Micro*scope (c, g, h, k): (a) Marchantiophyta, Conocephalum; (b) Rhodophyta, Phacelocarpus; (c) Glaucophyta, Cyanophora; (d) Scyphozoa, Chrysaora; (e) Choanoflagellata (f) Fungi, Agaricus; (g) Myxogastria, Dydimium (‘Hyperamoeba’); (h) Archeamoeba, Mastigina; (i) Myxogastria, Physarum; (d) (e) (f) (j) Stramenopile, Mallomonas; (k) Dinoflagellate, Polykrikos; (l) Rhizaria, Calcarina; (m) Metamonada, Trichonympha; (n) Discoba, Rapaza. (o) Metamonada, Kipferlia; (p) Cryptophyta, Chroomonas; (q) Cryptophyta, Cryptomonas; (r) Haptophyta, Gephyrocapsa. (g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)

cell wall around their plastids (Aitken and Stanier, 1979; tains a diverse assemblage of amoeboflagellates and slime Scott et al., 1984). Animals and fungi are both nested molds (Watkins and Gray, 2008; Shadwick et al., 2009) (Fig- within the Opisthokonta, which also includes choanoflagel- ures 1g–i). The Apusozoa branches as the nearest sister lates and chytrids, among other lineages (Lang et al., 2002) group to a clade consisting of the Opisthokonta and (Figures 1d–f). Most species in this supergroup possess a Amoebozoa (a clade that was once recognized as ‘uni- posterior flagellum that undulates to push the cell konts’; Kim et al., 2006; Cavalier-Smith and Chao, 2010). forwards (Cavalier-Smith and Chao, 2003a). The nearest Most of the supergroups also contain representatives of sister group to opisthokonts is the Amoebozoa, which con- what are arguably the three most dissimilar modes of

(eukaryotic) life on the planet: photoautotrophs, predators images of eukaryotic cells were taken with a transmission and parasites. For instance, the SAR clade contains plant-like electron microscope in the 1960s. The most recent review kelps and diatoms, saprophytic water molds (oomycetes), of the eukaryotic flagellar apparatus in microalgae was filter-feeding radiolarians, predatory ciliates and the notori- published more than a decade ago (Moestrup, 2000); how- ous blood-born parasite Plasmodium (Patterson, 1989; Cava- ever, a lot has changed since then, especially knowledge lier-Smith and Chao, 2003b; Leander and Keeling, 2003; about new lineages of eukaryotic cells and where they fit Roger and Simpson, 2009; Burki et al., 2010; Parfrey et al., into the overall tree of eukaryotes. In an attempt to under- 2010) (Figures 1j–l). The Excavata contains photosynthetic stand broad patterns of cytoskeletal homology in eukary- Euglena and relatives, the diarrhea-causing parasite Giardia, otes, we present a relatively comprehensive overview and several other different lineages of parasites and free-liv- of the eukaryotic flagellar apparatus within a modern ing phagotrophs (Figures 1m–o). There is still debate as to molecular phylogenetic context. We address the known whether or not the Excavata is monophyletic (a group that cytoskeletal diversity within the major groups of eukary- consists of an ancestor and all of its descendants), an infer- otes introduced above in order to infer ancestral traits and ence that happens to be crucial for understanding the origin subsequent trait changes during the evolutionary history and evolution of the eukaryotic cytoskeleton (Simpson and of the flagellar apparatus. Roger, 2004; Simpson et al., 2006). The synthesis of cyto- The eukaryotic flagellar apparatus is almost always com- skeletal traits we provide below suggests that the Excavata posed of two (or more) basal bodies that anchor the axo- is not monophyletic and instead represents a (paraphyletic) nemes, if present, and different microtubular roots; the stem group from which all other eukaryotes evolved. system functions as the MTOC for the entire eukaryotic cell (Moestrup, 1982; Sleigh, 1988; Andersen et al., 1991; Beech FUNDAMENTAL FEATURES OF MTOCS: THE FLAGELLAR et al., 1991; Moestrup, 2000) (Figure 2). The flagellar appa- APPARATUS ratus is therefore a complex ultrastructural system in Comparative morphology of the microtubular cytoskeleton almost every eukaryotic cell, and plays vital roles in a in eukaryotes started at about the same time the first variety of cell functions, such as locomotion, feeding

(a) (b) (c)

(d) (e)

Figure 2. Transmission electron microscopy (TEM) sections showing the divesity and major components of the flagellar apparatus in five representive lineages of eukaryotes: (a) Plantae, Pyramimonas (‘Prasinophyta’), modified with permission from Hori and Moestrup (1987); (b) Stramenopile, Apoikia (Stramenopile); (c) Excavata, Uteronympha (Metamonada), modified with permission from Brugerolle (2006b); (d) Haptophyta, Pleurochrysis, modified with permission from Inouye and Pienaar (1985); (e) Collodictyonidae, Collodictyon, modified with permission from Brugerolle et al. (2002). Scale bars: (a, b, d, e) 200 nm; (c) 400 nm.

(phagocytosis), and the formation of microtubular arrays THE DIVERSITY OF MTOCS REFLECTS THE MAJOR for structural support, vesicle transport and cell division GROUPS OF EUKARYOTES (Moestrup, 1982). The overall architecture of the flagellar Comparisons of MTOCs in different groups of eukaryotes is apparatus is relatively stable within major taxonomic complicated by the fact that researchers working in different groups, where only subtle differences in structure tend to fields (e.g. phycology versus protozoology versus zoology exist between closely related lineages (e.g. ‘family level’ versus botany) have applied different terms and notation to and ‘genus level’; Moestrup, 2000; Figures 3–6). The describe components of the flagellar apparatus in their flagellar apparatus represents one of the only ultrastructur- organsims of interest. In order to keep our homology al systems in eukaryotic cells that is essentially universally statements as clear as possible, we have applied the distributed, conserved enough to make confident homol- terminology recommended by Moestrup (2000) to describe ogy statements over large phylogenetic distances and vari- the flagellar apparatus of all the eukaryotic lineages high- able enough to discriminate different lineages from one lighted below. another. Any attempt to reconstruct the evolutionary history of Plantae (green algae, land plants and relatives) the flagellar apparatus requires confident homology state- With a few exceptions, members of this clade have a cruci- ments about the relevant traits being compared in differ- ate flagellar apparatus, or some modification thereof (Fig- ent eukaryotes. Therefore, it is important to identify which ures 3a–d). The basic apparatus consists of two basal basal bodies, flagella and microtubular roots are compara- bodies (B1 and B2), often in a V-like orientation, and four ble between the species of interest. This is not always microtubular roots (R1–R4). The traditional notations for straightforward. In Chlamydomonas, for instance, two fla- the flagellar apparatus in green algae (1d, 1s, 2d, 2s or X-2- gella of the same length are inserted in the apical region X-2) are revised here to R1, R2, R3 and R4, respectively. B2 of the cell; based on gross morphology alone it is impos- and its associated R3 and R4 form a unit that is develop- sible to distinguish the two flagella in the right or left ori- mentally equivalent to B1 and its associated R1 and R2, entation of the cell. However, the two basal bodies (and but with a 180° rotation (Figure 3a). A distinctive multilay- associated flagella and microtubular roots) in the cell have ered structure (MLS) of variable size is connected to R1 in different generational origins (Gely and Wright, 1986; certain ‘prasinophytes’, in the zoospores and sperm of Beech et al., 1988, 1991; Moestrup, 2000). One basal body streptophyte algae, and in the sperm of most non-flower- is younger (basal body 2 or B2), and is formed anew during land plants (Moestrup, 1978; Stewart and Mattox, 1978; ing the most recent round of cell division; the other basal Melkonian, 1980, 1982; O’Kelly and Floyd, 1983; Vouilloud body is older (basal body 1 or B1), and is formed during et al., 2005) (Figures 3b–d). Red algae lack a flagellar an earlier round of cell division and is inherited from the apparatus altogether, which is highly unusual for eukary- parent cell. Therefore, the basal bodies and associated fla- otes, and knowledge of the glaucophyte flagellar apparatus gella in each cell reflect different generations (or cell divi- is currently incomplete (Mignot et al., 1969; Kies, 1979, sion events), and are inherited in a semi-conservative 1989). pattern much like DNA replication. If we know which of the two basal bodies is oldest (B1), then we can deter- Opisthokonta (animals, fungi and relatives) mine a right or left orientation of the cell and identify the With a few exceptions, members of this clade possess four different microtubular roots (R1, R2, R3 and R4) asso- MTOCs consisting of two orthogonal basal bodies (syn., ciated with the basal bodies (Figure 3a). R1 and R2 are centrioles) within an amorphous matrix (i.e. the centro- associated with the older B1; R3 and R4 are associated some) and a system of radiating microtubules (Figures 3e– with the younger B2. The microtubular roots are also dis- h). The centrioles within the centrosome of animal cells tinguished from one another using their relative positions are structurally homologous to basal bodies, but no longer in the cell, their point of insertion onto the basal bodies, organize flagellar axonemes. There are no conspicuous and their affiliations with other cell features. In green microtubular roots present in this lineage (Hibberd, 1975; algae, for instance, the positions of the eyespot and the Barr, 1981; Karpov and Leadbeater, 1998). mating structure are closely associated with R4 and R2, respectively (Beech et al., 1988, 1991) (Figure 3a). This Amoebozoa approach allows us to identify homologous structures in the eukaryotic flagellar apparatus that can be compared With a few exceptions, members of this clade have three across the tree of eukaryotes; however, reconstructions or four robust microtubular roots linked to two basal and subsequent comparisons of the flagellar apparatus bodies. B1 anchors R1 and R2, the latter of which is split are challenging, because these studies require sophisti- into an inner ribbon (iR2) and an outer ribbon (oR2). R3 is cated microscopy, spatial reasoning and the consistent connected to B2 and functions to anchor a dorsal array of usage of detailed terminology. superficial microtubules (Figures 3i–l). Some amoboezo-

(a) (b) R4 (2s) (c) (d) 4 2 1 R3 (2d) R4 2 1 R1 MLS 2 MLS 3 (2s) (1d) 1 2 1 R4 MLS R1 R1 R3 Plantae R2 (1d) R2 R2 R3 R2 (1s) R1 (2d) (1s)

Chlamydomonas Pterosperma Coleochaete (e) (f) (g) (h)

2 2 1 2 1 2 1 1 Opisthokonta

Gallus Codosiga Monoblepharella

(i) (j) (k) (l)

2 2 2 2 R3 R3 R3? 1 R3 1

oR2 iR2 R1 Amoebozoa iR2 oR2 R1 oR2 Dydimium Ceratiomyxella Mastigina iR2

Figure 3. Illustrations of the flagellar apparatus in the Plantae, Opisthokonta and Amoebozoa. The diversity of the flagellar apparatus within each group of eukaryotes is represented by a row of three different genera and a simpler depiction of the inferred ancestral traits for the group (right-hand column, with darker background). The labels in parentheses (1d, 1s, 2d and 2s) in (a) and (b) refer to the traditional terminology in the Plantae. Arrows indicate the directions of the flagella. (a) Chlorophyta, Chlamydomonas reinhardtii, redrawn based on the data in Ringo (1967) and Geimer (2004). (b) ‘Prasinophyta’, Pterosperma cristatum, redrawn based on the data in Inouye et al. (1990). (c) Streptophyta, Coleochaete pulvinata, redrawn based on the data in Sluiman (1983). (d) The inferred ancestral traits for the flagellar apparatus in the Plantae. (e) Vertebrate interphase cell of Gallus, redrawn based on the data in Doxsey (2001). (f) Choanoflagellate, Codosiga botrytis, redrawn based on the data in Hibberd (1975). (g) Fungus, Monoplepharella, redrawn based on the data in Barr (1978, 1981). (h) The inferred ancestral traits for the flagellar apparatus in the Opisthokonta. (i) Myxogastria, Dydimium dachnaya, redrawn based on the data in Walker et al. (2003). (j) Schizoplasmodiida, Ceratiomyxella, redrawn based on the data in Spiegel (1981). (k) Archamoebae, Mastigina hylae, redrawn based on the data in Brugerolle (1991). (l) The inferred ancestral traits for the flagellar apparatus in the Amoebozoa. ans, like most mastigamoebids and uniflagellate myxoga- alent to what we have already recognized as the MLS in strid slime molds, have lost B1 and its associated microtu- other taxa (e.g. the Plantae, Figures 3b–d). The I fiber bular roots (Brugerolle, 1991; Karpov and Myl’nikov, 1997; has a cross-hatched appearance and is ventrally located Walker et al., 2003) (Figure 3k). on the concave side of R2. The separation of R2 into iR2 and oR2 ribbons supports the two sides of a ventral Excavata feeding groove (Simpson and Patterson, 2001; Simpson, The most complicated flagellar apparatus known is com- 2003; Simpson and Roger, 2004; Yubuki et al., 2007, mon in a variety of tiny (<10 lm) bacterivorous 2013a) (Figures 1o and 4a–d). excavates (Figures 1o and 4a–d). B1 not only anchors Stramenopiles R1, SR and R2, split into iR2 and oR2, but also four dis- tinct fibers: A, B, C and I fibers. B2 anchors R3, which Members of this clade are extremely diverse in morphol- supports a dorsal fan of superficial microtubules (Fig- ogy and size (ranging from a few lm in some flagellates ures 4a,b), and in some taxa (e.g. Andalucia), R4 to over 50 m in kelps), and have diverse modes of nutri- extends from the posterior side of B2 within the space tion, including phototrophy, mixotrophy, phagotrophy, between B1 and B2 (Figures 4c,d). The C fiber is osmotrophy and parasitism. Despite this diversity, the attached to the dorsal side of R1 and is therefore equiv- structure of the flagellar apparatus has remained remark-

(a) 2 (b) 2 (c) 2 (d) A 2 I F F F A R3 B A 1 I R4 I 1 B 1 R4 MLS R3 1 A MLS R3 MLS MLS B I

Excavata B iR2 oR2 iR2 SR iR2 SR SR oR2 R1 R1 oR2 iR2SR R1 R1 oR2 Carpediemonas Malawimonas Andalucia

(e) 2 (f) R3 R4 (g) 2 (h) R3 2 R3 2 1 R4 1 1

1 R1 oR2 R3 R1 R4 Stramenopile iR2 iR2 oR2 R1 oR2 iR2SR R1 oR2 iR2 Rictus SR Thraustrochytrium Apoikia

R3 (i) (j) R3 (k) (l) R3? R3? R4? 2 2 2 2? R4 1 R1 1 1 R1? R2 R4 Rhizaria 1? R2? R2? R4? R1? R2 R1 Heteromita Bigelowiella Polymyxa

Figure 4. Illustrations of the flagellar apparatus in the Excavata, Stramenopiles and Rhizaria. The diversity of the flagellar apparatus within each group of eukaryotes is represented by a row of three different genera and a simpler depiction of the inferred ancestral traits for the group (right-hand column, with darker background). Arrows indicate the directions of flagella. (a) Metamonada, Carpediemonas membranifera, redrawn based on the data in Simpson and Patterson (1999). (b) Malawimonas jakobiformis, redrawn based on the data in O’Kelly and Nerad (1999). (c) Discoba, Andalucia (Jakoba) incarcerata, redrawn based on the data in Simpson and Patterson (2001). (d) The inferred ancestral traits for the flagellar apparatus in the Excavata. (e) Bicosoecida, Rictus lutensis, redrawn based on the data in Yubuki et al. (2010). (f) Labyrinthulomycetes, Thraustochytrium aureum, redrawn based on the data in Barr and Allan (1985). (g) Chrysophyceae, Apoikia lindahlii, redrawn based on the data in Kim et al. (2010). (h) The inferred ancestral traits for the flagellar apparatus in the Stramenopile. (i) Heteromita sp., redrawn based on the data in Karpov (1997). (j) Chlorarachniophyta, Bigelowiella natans, redrawn based on the data in Moestrup and Sengco (2001). (k) Endomyxa, Polymxa graminis, redrawn based on the data in Barr and Allan (1982). (l) The inferred ancestral traits for the flagellar apparatus in the Rhizaria. ably uniform throughout the clade (Patterson, 1989; Andersen, niferans, chlorarachniophytes, testate amoebae and sev- 1991, 2004; Moestrup and Andersen, 1991; Moestrup, eral kinds of predatory amoeboflagellates), and has 2000). B1 anchors R1 and R2, and as in excavates, the sep- therefore been established on the basis of comparative aration of R2 into iR2 and oR2 ribbons supports the two genomic data rather than shared morphological traits. sides of a ventral feeding apparatus (Figures 4e–h). In Nonetheless, flagellated members of this clade possess early-diverging stramenopiles, like the bicosoecid Rictus, the basic elements of the eukaryotic flagellar apparatus: B1 B1 also anchors a distinctive root formed from a single anchoring R1 and R2, and B2 anchoring R3 and R4 (Kar- microtubule (SR) that is positioned between R1 and R2 pov, 1997; Moestrup and Sengco, 2001; Cavalier-Smith and (Moestrup and Thomsen, 1976; Karpov et al., 2001; Yubuki Chao, 2003b). R2 is not split into two ribbons, and no SR, et al., 2010) (Figure 4e). B2 anchors R3 and R4, the former MLS, or a superficial array of microtubules associated with of which supports an array of superficial microtubules R3 is present (Figures 4i–l). (Andersen, 1991; Kim et al., 2010). Alveolata Rhizaria Members of this clade share several ultrastructural traits This clade consists of many different lineages that are (e.g. an arrangement of alveoli beneath the plasma mem- extremely diverse in morphology (e.g. radiolarians, forami- brane and distinctive micropores), despite the very high

(a) (b) R4 (c) (d) R3 R3 R3? R3 R4 2 2 2 2 R1

R2 1 1 1 R2?

Alveolata R1 1 R2 R1? R2 R1 Dexiotricha Gymnodinium Colpodella (e) (f) (g) (h) R3 R3 R3 2 R4 R4 R4 H 2 2 H H 2 1 H R2 1 oR2 1 1 iR2? R1 Haptophyta R2

R1 oR2 iR2 R1 Pavlova Chrysoculter R1 Pleurochrysis

(i) (j) (k) R4 (l) R3 R3 R3 R4 2 R4 2 2 2 R4 R3 1 1 R1 1 R1 oR2 1 MLS oR2 iR2 R1 iR2? R2 R2

Cryptophyta & Katablepharida Cryptomonas Goniomonas Katablepharis

Figure 5. Illustrations of the flagellar apparatus in the Alveolata, Haptophyta and Cryptophyta. The diversity of the flagellar apparatus within each group of eukaryotes is represented by a row of three different genera and a simpler depiction of the inferred ancestral traits for the group (right-hand column, with darker background). Arrows indicate the directions of flagella. (a) Ciliate, Dexiotricha redrawn based on the data in Peck (1977). (b) Dinoflagellate, Gymnodinium chlorophorum, redrawn based on the data in Hansen and Moestrup (2005). (c) Colpodelida, Colpodella vorax, redrawn based on the data in Brugerolle (2002b). (d) The inferred ancestral traits for the flagellar apparatus in the Alveolata. (e) Pavlovophyveae, Pavlova pinguis, redrawn based on the data in Green (1980). (f) Prymnesiophyceae, Chrysoculter rhomboideus, redrawn based on the data in Nakayama et al. (2005). (g) Prymnesiophyceae, Pleurochrysis sp. (coccolithophorid), redrawn based on the data in Inouye and Pienaar (1985). (h) The inferred ancestral traits for the flagellar apparatus in the Haptophyta. (i) Cryptophyceae, Cryptomonas ovata, redrawn based on the data in Perasso et al. (1992). (j) Goniomonadea, Goniomonas avonlea, redrawn based on the data in Kim and Archibald (2013). (k) Katablepharida, Katablepharis sp., redrawn based on the data in Lee et al. (1992). (l) The inferred ancestral traits for the flagellar apparatus in the Cryptophyta. levels of diversity represented in the three major subc- Roberts and Roberts, 1991; Brugerolle, 2002a,b; Myl’nikova lades: dinoflagellates, ciliates and apicomplexans. The and Myl’nikov, 2010). structure of the flagellar apparatus in these groups and in Haptophyta flagellates such as Colpodella and perkinsid zoospores is also very diverse, which makes it difficult to infer the unify- This group of microalgae is unfied by a novel cell exten- ing traits of the alveolate MTOC (Figures 5a–d). For sion called the haptonema (H), and consists of two main instance, apicomplexans are obligate intracellular prata- subclades: the Prymnesiophyceae (including coccolitho- sites that have essentially lost the flagellar apparatus phorids) and Pavlovophyceae (Figure 1r). In prymnesio- altogether (except in some microgametes), whereas cili- phyceans (e.g. Chrysochromulina, Chrysoculter and ates have duplicated the flagellar apparatus to extreme lev- Pleurochrysis), each basal body usually posesses two els, often organizing hundreds of copies over the cell microtubular roots. In coccolithophorids (Pleurochrysis), surface. Nonetheless, all four roots, R1–R4, associated with R1 and R2 organize robust crystalline arrays of microtu- two basal bodies, B1 and B2, are present in at least some bules (Figure 5g). B2 anchors R3 and R4, which consist of members of the Alveolata (Figures 5a–d). Moreover, an only a few microtubules (Beech et al., 1988; Birkhead and array of superficial microtubules extends from R3 in some Pienaar, 1995; Inouye and Pienaar, 1985; Kawachi and Ino- alveolates, especially in dinoflagellates (Roberts, 1991; uye, 1994; Yoshida et al., 2006) (Figures 5f–g). In pavlovo-

(a) (b) (c) R3 (AS) (d) 2 2 2 R4 (AR) R3 2 R3 1 R3 (DL) R4 oR2 1 1 1

(RM)

Apusozoa oR2 (RH) oR2 SR (L3/CTM) R1 SR R1 (PM) iR2 (L2) oR2 iR2 R1 (L1) iR2 SR R1 (LM)(PM) Apusomonas iR2 SR Thecamonas Ancyromonas

R3 (e) (f) R3 (g) (h) R3 2 2 2 2 3 1 RR 1 4 1 R3 1 LR R1

LF R2 iR2 RF iR2 Collodictyonidae oR2 RF LF R1 RF R1 R2 LF oR2 R1 Sulcomonas Collodictyon Diphylleia

Figure 6. Illustrations of the flagellar apparatus in the Apusozoa and Collodictyonidae. The diversity of the flagellar apparatus within each group of eukaryotes is represented by a row of three different genera and a simpler depiction of the inferred ancestral traits for the group (right-hand column, with darker background). Arrows indicate the directions of flagella. (a) Apusomonada, Apusomonas proboscidea, redrawn based on the data in Karpov (2007). (b) Apusomonada, Thecamonas trahens, redrawn based on the data in Heiss et al. (2013). (c) Ancyromonada, Ancyromonas sigmoides, redrawn based on the data in Heiss et al. (2011). Note that Heiss et al. (2011) designated the anterior singlet (AS) and the anterior root (AR) of Ancyromoans as R4 and R3, which shgould be reversed as R3 and R4, respectively, based on the locations and directions of their origin on the anterior basal body. (d) The inferred ancestral traits for the flagellar apparatus in the Apusozoa. (e) Collodictyonidae, Sulcomonas lacustris, redrawn based on the data in Brugerolle (2006a). (f) Collodictyonidae, Collodictyon triciliatum, redrawn based on the data in Brugerolle et al. (2002). (g) Collodictyonidae, Diphylleia rotans (=Aulacomonas submarina), redrawn based on the data in Brugerolle and Patterson (1990). (h) The inferred ancestral traits for the flagellar apparatus in the Collodictyonidae. phyceans, B1 anchors R1 and R2, the latter of which is sep- members of the Apusozoa based on phylogenetic infer- arated into two ribbons; B2 lacks microtubular roots (R3 ences derived from small subunit rRNA gene sequences and R4) altogether (Green, 1980) (Figures 5e–h). and the presence of a theca (Cavalier-Smith and Chao, 2003a). The overall architecture of the flagellar apparatus is Cryptophyta and Katablepharida complicated, and is most similar to the flagellar apparatus Members of these groups (Figures 1p–q) constitute a rela- in excavates (e.g. Carpediemonas and Malawimonas) and tivley unified clade of microalgae that possess a flagellar stramenopiles (e.g. Rictus and Apoikia). B1 anchors R1, SR apparatus with two basal bodies, B1 and B2, and four and R2, and the separation of R2 into the iR2 and oR2 rib- microtubular roots, R1–R4. The previously recognized stri- bons supports the two sides of a ventral feeding apparatus ated root-associated microtubules (SRm) are inserted on (Figures 6a–d). An MLS attached to R1 is not present. B2 the dorsal side of B1, and are therefore recognized here as anchors R3 and sometimes an R4 (e.g. Ancyromonas), the R1 (Figures 5i–l). The previously recognized ‘rhizostyle’ is a former of which supports an array of superficial microtu- bundle of microtubules running longitudinally from the bules (Figures 6c,d). ventral side of B1, and is therefore recognized here as R2 Collodictyonidae (Moestrup, 2000). The band of microtubules that inserts on the dorsal side of B2 is recognized here as R3; the band of This is a small but distictive group of microbial eukaryotes microtubules that inserts on the ventral side of B2 is recog- with flagellar apparatuses that are reminiscent of those nized here as R4 (Roberts, 1984; Perasso et al., 1992; Kim found in excavates, apusomonads and stramenopiles. B1 and Archibald, 2013) (Figure 5i–l). In early diverging cryp- anchors R1 and R2, and the separation of R2 into the iR2 tomonads, like Goniomonas, and katablepharids, the and oR2 ribbons supports the two sides of a ventral equivalent of the MLS described in other taxa is present feeding apparatus (Figures 6e–h). An MLS attached to R1 (Kim and Archibald, 2013; Lee et al., 1992) (Figure 5k). is not present. B2 anchors R3, which supports an array of superficial microtubules. There is no R4 or SR associated Apusozoa with B2 (Brugerolle, 2006a; Brugerolle and Patterson, The monophyly of apusomonads and ancyromonads is still 1990; Brugerolle et al., 2002; Cavalier-Smith and Chao, somewhat controversial, but we consider ancyromonads 2010). The so-called left and right fibers (LF and RF) are

© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 75, 230–244 238 Naoji Yubuki and Brian S. Leander associated with R1 and R2, respectively, and support the cell division, and the complete transformation of B2 and left and right margins of the ventral feeding groove (Fig- its roots to B1 and its roots. Nonetheless, the presence ures 6e–h). Two extra basal bodies, B3 and B4, are pres- of an MLS is a trait shared by chlorophytes, strepto- ent in the tetraflagellate Collodictyon; B3 and B4 anchor phytes and almost certainly the most recent ancestor of the left root (LR) and the right root (RR), respectively, both green plants (i.e. the Viridiplantae; Lewis and McCourt, of which are the developmental equivalent of R2 in the 2004; McCourt et al., 2004). next generation. The MLSs are also more broadly distributed across the tree of eukaryotes, such as in excavates, cryptophytes, Breviata katablepharids, Palpitomonas and some dinoflagellates This genus includes only one species, Breviata anathema, (Wilcox, 1989; Roberts and Roberts, 1991; Lee et al., 1992; with an unresolved phylogenetic position within the tree of Yabuki et al., 2010; Kim and Archibald, 2013) (Figure 7). eukaryotes. These cells have four robust microtubular The MLS has been labeled as the C fiber in excavates. roots linked to two basal bodies. B1 anchors R1 and R2, The C fiber in some excavates, like jakobids, is very well the latter of which is split into an inner ribbon (iR2) and an developed, and the potential homology of the C fiber with outer ribbon (oR2). A distinctive singlet root (SR) is posi- the MLS in green plants has been discussed previously tioned between R1 and R2. R3 is connected to B2 and func- (O’Kelly, 1993). Furthermore, a remnant of the B fiber in tions to anchor a dorsal array of superficial microtubules some excavates, like Dysnectes and Kipferlia, forms a thin (Walker et al., 2006; Minge et al., 2009). sheet-like structure on the ventral side of R1 (Yubuki et al., 2007, 2013a). This structure in excavates is very similar in HOMOLOGOUS ELEMENTS IN DIFFERENT MTOCS form and position to the ‘plate-like structure’ described in the prasinophyte Crustomastix and the ‘keels’ described Previous reconstructions and comparisons of the flagellar in the prasinophyte Mesostigma (Melkonian, 1989; Nakay- apparatus were accomplished with a relatively limited ama et al., 2000). A green algal-like MLS has also been comparative context, and accordingly these studies estab- reported in other excavates like the euglenozoan Eutrepti- lished different descriptive terms for the MTOCs in differ- ella (Moestrup, 1978, 1982). Altogether, these data provide ent groups of eukaryotes. Detailed information about the evidence that R1 and an associated MLS is homologous excavate flagellar apparatus, for instance, was not avail- in plants and excavates, as well as in cryptophytes and able until relatively recently (Yubuki et al., 2013a). Our katablepharids, suggesting that these structures were synthesis suggests that the basic architecture of the already present in the most recent common ancestor of eukaryotic flagellar apparatus consists of two basal eukaryotes (Figure 7). bodies (B1 and B2), four main microtubular roots (R1–R4) and a singlet root originating from B1 (Moestrup, 2000). R2 facilitates phagotrophy The microtubules originating from R2 form a feeding appa- R1 and associated MLS ratus on the ventral side of the cell in several different When present, R1 originates from the left side of B1 and groups of protists (Patterson, 1989; Andersen, 1989, 1991; extends towards the left side of the cell. R1 is associated Simpson, 2003; Simpson and Roger, 2004; Heiss et al., with an MLS in the flagellated stages of streptophyte life- 2011, 2013; Yubuki et al., 2009, 2013b). In ‘typical’ exca- cycles (Coleochaete, Chara, Klebsormidium and the vates, phagotrophy is accomplished between two separate sperm of liverworts, mosses, ferns, Ginkgo and cycads), ribbons of microtubules derived from R2: oR2 and iR2 (an and in some prasinophytes (e.g. Cymbomonas, Halosph- outer ribbon and an inner ribbon; Bernard et al., 1997; aera and Pterosperma), which are inferred to have Simpson et al., 2000; Yubuki et al., 2007, 2013a). Euglen- retained many ancestral states for the Plantae as a whole ozoans, by contrast, are a very diverse group of atypical (Carothers and Kreitner, 1967; Moestrup, 1978; Melkonian, excavates with a different kind of feeding apparatus con- 1980; Li et al., 1989; Graham and Wilcox, 2000; Vouilloud sisting of two robust feeding rods that function together et al., 2005; Archibald, 2009). Interestingly, an early with a system of four vanes in euglenids (Leander et al., diverging streptophyte, Mesostigma viride, has two 2007). Euglenozoans lack many conserved traits associated MLSs, one associated with R1 and one associated with with the excavate flagellar apparatus and feeding groove, R3 (Rogers et al., 1981). R1 and R3 are developmentally but the group is strongly affiliated with the excavate con- equivalent after flagellar transformation, whereby the R3 cept through their robust molecular phylogenetic relation- on B2 in the parent cell transforms into the R1 on B1 in ship with jakobids (Simpson, 2003; Simpson and Roger, one of the daughter cells after division (Moestrup, 2000). 2004). Even though the feeding apparatus in euglenozoans The presence of an MLS on R3 in M. viride is inferred to seems fundamentally different from that in typical exca- reflect heterochrony, namely a change in developmental vates, the microtubules that support the feeding rods still timing so that the MLS–R1 association develops prior to originate from R2 (synonymous with the ‘ventral root’ in

Stramenopile

Rhizaria Alveolata Excavata

Plantae

Apusozoa SAR Haptophyta

2 R3

R4 Cryptophyta Opisthokonta 1 MLS

oR2 R1 iR2 SR Collodictyonidae

Amoebozoa ?

R1 with an MLS An array of superficial R2 separated into microtubules on R3 oR2 and iR2 R4 SR

Figure 7. Illustrations of the inferred ancestral traits for the flagellar apparatus in all 11 eukaryotic groups mapped onto a modern molecular phylogenetic context. The five traits mapped onto the tree are represented by different shapes: microtubule root 1 (R1) with a multilayered structure (MLS), R2 involved with phagotrophy, R3 with an array of superficial microtubules, a singlet root (SR) that helps support the ventral feeding groove and R4. Solid shapes refer to the presence of a trait; dashed outlines of shapes refer to the loss of a trait. The inferred ancestral flagellar apparatus for the most recent ancestor of eukaryotes is best represented by the traits found in typical excavates. The most recent common ancestor of the Apusozoa, Opisthokonta and Amoebozoa lost an MLS on R1; R4 was lost in the most recent common ancestor of the Opisthokonta and Amoebozoa. An MLS on R1 was also lost in the most recent common ancestor of the SAR (Stramenopiles, Alveolata and Rhizaria) clade. euglenozoan notation; Attias et al., 1996; Leander, 2004; ventral face of oR2 in some stramenopiles is essentially Yubuki et al., 2013b). identical to the position and appearance of the I fiber, pres- The functional relationship of R2 microtubules with ent in different lineages of excavates, and a fibrous struc- feeding structures extends to several other groups of ture assciated with oR2 in apusomonads (Karpov, 2007; eukaryotes, such as stramenopiles, amoebozoans, apuso- Heiss et al., 2011; Yubuki et al., 2010). zoans and collodictyonids. Detailed investigations of Collodictyonids (Collodictyon, Diphylleia and Sulcomon- stramenopile feeding behavior in chrysophycean algae as) also have a robust ventral groove that runs down the (e.g. Epipyxis and Apoikia) and deep-branching heterotro- longitudinal axis of the cell that functions to phagocytize phic bicosoecids (e.g. Rictus) demonstrate that prey relatively large food particles. This groove is supported by particles (i.e. bacteria) are engulfed via a ‘cytostome’ that microtubules derived from both R1 and R2, and highly is formed by inner and outer microtubules derived from R2 resembles the feeding grooves of excavates and stra- (Andersen and Wetherbee, 1992; Wetherbee and Andersen, menopiles (Brugerolle and Patterson, 1990; Brugerolle 1992; Kim et al., 2010; Yubuki et al., 2010). The separation et al., 2002; Brugerolle, 2006a). The flagellar apparatus of of R2 into two ribbons, oR2 and iR2, is widely distributed excavates and collodictyonids has been compared in in stramenopiles, and is considered a shared feature for detail, and the so-called right ventral root (rvR) and Golgi the entire clade (Andersen, 1991; Moestrup and Andersen, nucleus root (gnR) of Sulcomonas (Brugerolle, 2006a) is 1991). It is worth noting that cross-hatched fibers on the inferred to be homologous with the oR2 and iR2 in exca-

© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 75, 230–244 240 Naoji Yubuki and Brian S. Leander vates (Cavalier-Smith and Chao, 2010). The iR2 (synony- array of superficial microtubules that shape the cell sur- mous with gnR) in collodictyonids extends towards the face. In excavates, this array of superficial microtubules is interior of the cell, instead of supporting the superficial called the ‘dorsal fan’ (O’Kelly, 1993; O’Kelly and Nerad, feeding groove itself (as in excavates and stramenopiles), 1999; Simpson and Patterson, 1999; Simpson, 2003; Simp- which facilitates the engulfment of larger prey particles son and Roger, 2004). R3 in euglenids (corresponding to such as other eukaryotic cells (Cavalier-Smith and Chao, the ‘dorsal root’) organizes an array of microtubules, called 2010). the ‘dorsal band’, that ultimately supports the complex and The R2 in amoebozoans (e.g. Didymium), haptophytes highly distinctive system of pellicle strips (Owens et al., (e.g. Pavlova and Diacronema) and the cryptomond Gonio- 1988; Yubuki et al., 2009; Yubuki and Leander, 2012). This monas is also separated into an oR2 and iR2; however, suggests that the novel flagellar apparatus in englenozo- whether or not these microtubules function in feeding is ans contains several homologous traits with the basic fla- unknown (Green and Hibberd, 1977; Green, 1980; Walker gellar apparatus found in typical excavates. et al., 2003). The phagotrophic alveolate Colponema lox- In stramenopiles, R3 (confusingly corresponding to R1 odes possesses a ventral feeding groove that is supported in earlier literature) curves clockwise towards the anterior by two microtubular roots that correspond with R2, a con- end of the cell and anchors the superficial microtubules figuarion that resembles the MTOCs in excavates and that support the cell shape: this configuration is a unify- collodictyonids (Mignot and Brugerolle, 1975; Myl’nikova ing feature of the group as a whole, ranging from a and Myl’nikov, 2010). However, ultrastructural studies of diverse assemblage of photosynthetic species to tiny bac- this flagellate are incomplete, so compelling homology teriphagus flagellates (Andersen, 1987, 1991; Moestrup statements about the MTOC in Colponema would be pre- and Andersen, 1991; Karpov et al., 2001; Kim et al., 2010; mature. Although it is possible that similarities in the form Yubuki et al., 2010). A very similar configuration of R3 and function of R2 microtubules could reflect convergent microtubules has been described in dinoflagellates (Alve- evolution, there is no supporting evidence for this. The olata), apusozoans, amoebozoans and collodictyonids most parsimonious interpretation of current data is that (Farmer and Roberts, 1989; Brugerolle and Patterson, the separation of R2 microtubules into oR2 and iR2 ribb- 1990; Roberts, 1991; Roberts and Roberts, 1991; Spiegel, bons that support a feeding apparatus represents deep 1991; Brugerolle et al., 2002; Walker et al., 2003; Karpov, homology that evolved in the most recent common ances- 2007). tor of excavates, stramenopiles, apusozoans, amoebozo- R4 ans, collodictyonids, haptophytes, cryptophytes (e.g. Goniomonas) and probably alveolates. The presence of R4 is widely distributed across the tree of eukaryotes, absent only in opisthokonts, amoebozoans The singlet root and collodictyonids. This root forms a band of only a few This ‘root’ is formed by a single microtubule with a distinc- microtubules (less than five) that originate from the ventral tive orientation within the overall context of the eukaryotic side of B2 and extend towards the left side of the cell. flagellar apparatus. Although it is identifiable in many dif- Although R4 is common in eukaryotes, its function is not ferent groups of eukaryotes, it is inconspicuous and easily well understood. In stramenopiles, the microtubules of R4 overlooked. Nonetheless, the singlet root (SR) is relatively support the left margin of the feeding groove (Kim et al., well studied in excavates, and is considered an important 2010; Yubuki et al., 2010). It is important to realize that R4 trait that unifies the entire group (Simpson, 2003; Simpson is developmentally equivalent to R2 (on B1); during cell and Roger, 2004). In excavates, stramenopiles (e.g. bico- division, B2 with R4 (and R3) in the parent cell transforms soecids) and apusozoans, the SR originates on B1 and into B1 with R2 (and R1) in one of the daughter cells extends towards the posterior end of the cell, between R1 (Moestrup, 2000; Yubuki and Leander, 2012; Yubuki et al., and R2 microtubules, to support the feeding apparatus 2013a). Therefore, the presence of R4 microtubules facili- (Moestrup and Thomsen, 1976; Karpov et al., 2001; Yubuki tates the future development of the critical R2 microtubules et al., 2010; Heiss et al., 2011, 2013). Athough the SR has that ultimately support the ventral feeding apparatus in not been fully investigated in all major groups of eukary- many groups of eukaryotes. otes, the presence of this root in excavates, apusozoans and RECONSTRUCTING THE ANCESTRAL MTOC stramenopiles suggests that it is a homologous trait derived from the most recent common eukaryotic ancestor. The flagellar apparatus of typical excavates has all of the traits present in various combinations in other major R3 and arrays of superficial microtubules groups of eukaryotes: R1 with an MLS; R2 involved with Several major groups of eukaryotes have an R3 that origi- phagotrophy; R3 with an array of superficial microtubules; nates from basal B2 and curves clockwise towards the SR that helps support the ventral feeding groove; and R4. anterior end of the cell. R3 functions as the MTOC for an In other words, the excavate flagellar apparatus is a sum-

© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 75, 230–244 Macroevolution of microtubule organizing centers 241 mation of all of the major components of the flagellar dsitributed across the tree of eukaryotes than is currently apparatus found in different lineages across the tree of appreciated (Figure 7). eukaryotes (Figure 7). Several of the phylogenetic relation- In fact, the major groups of eukaryotes all have a flagel- ships between the major clades of eukaryotes (i.e. the lar apparatus with a combination of components that can deepest nodes in the tree) are still uncertain, and this be traced back to the excavate configuration. This suggests context is ultimately needed to trace the origins of cellular that the five main components of the flagellar apparatus, traits back to the most recent ancestor of all eukaryotes when present, are homologous across the tree of eukary- (Figure 7). Nonetheless, the integration of molecular phylo- otes, and ultimately descended from an ‘excavate-like’ genetic data with comparative ultrastructural data from common ancestor (Figure 7). This hypothesis: (i) postu- diverse groups of protists has elucidated many events in lates that excavates are not monophyletic unless you syn- the evolutionary history of the eukaryotic cell. As explained onymize the group with the Eukarya, and (ii) leaves open a more below, the comparative analysis of the flagellar appa- very intriguing question – how did the complicated flagel- ratus presented here suggests that the flagellar apparatus lar apparatus of typical excavates evolve in the first place? found in several living excavates are fantastic examples of BROAD PATTERNS OF MTOC EVOLUTION: INDEPENDENT morphostasis that approximate the flagellar apparatus STREAMLINING present in the most recent ancestor of all eukaryotes (Figure 7). The diversity of the flagellar apparatus across the tree of The monophyly of the Excavata has so far not been eukaryotes is best explained by several independent losses supported in molecular phylogenetic analyses (Simpson, of ancestral, excavate-like traits (Figure 7). For instance, 2003; Berney et al., 2004; Simpson and Roger, 2004; Par- the most recent ancestor of opisthokonts (including ani- frey et al., 2006; Simpson et al., 2006; Ishida et al., 2010). mals and fungi) appears to have lost every microtubular The composition of this putative clade is grounded root present in the flagellar apparatus of the ancestral squarely on comparative ultrastructure and the punctate eukaryote, retaining only basal bodies B1 and B2 (Roger phylogenetic distribution of tiny flagellates with a remark- and Simpson, 2009) (Figure 7). ably complex and uniform flagellar apparatus and feeding The most recent ancestor of the Plantae, however, shares groove (i.e. the typical excavate cytoskeletal configura- R1–MLS and iR4 with excavates, but lost the SR, a tion). The Excavata consists of three major lineages that branched R2 (oR2 and iR2) and an array of superficial are otherwise very different from one another at both the microtubules from R3. The loss of SR, the branched R2 and ultrastructural and genomic levels, yet each contain the the associated feeding apparatus in the Plantae is almost typical excavate flagellar apparatus: (i) Discoba (Jakobida, certainly correlated with the origin of photosynthesis via Heterolobosea and Euglenozoa), (ii) Malawimonas, and primary endosymbiosis; this event changed the mode of (iii) Metamonada (Trimastix, Preaxostyla, Parabasalia and nutrition dramatically, which led to new selection pressures Fornicata) (Simpson, 2003; Simpson and Roger, 2004; that ultimately shaped the evolution of a more streamlined Simpson et al., 2006; Kolisko et al., 2010). The high level cytoskeleton. By contrast, the most recent ancestor of stra- of homology between the typical excavates in each of menopiles shares every component of the flagellar appara- these otherwise very different lineages provides the pri- tus with excavates, except for the loss of an MLS on R1. mary basis for the hypothesis that they are all part of a Although many stramenopiles are photosynthetic via sec- monophyletic group: the Excavata. ondary endosymbiosis (e.g. diatoms and brown algae), the As described above, however, the flagellar apparatus of most recent ancestor of the clade was probably a tiny bac- typical excavates is very similar to the flagellar apparatus terivore with a mode of nutrition that is nearly identical to in stramenopiles, apusozoans and collodictyonids (Fig- typical excavates. This helps explain why the overall flagel- ure 7). In all of these groups the microtubules of R1 and lar apparatus is so similar in excavates and stramenopiles, the two ribbons of R2 (oR2 and iR2) originate from B1 and an interpretation that applies to the relativley similar flagel- reinforce the feeding groove; R3 originates from the dorsal lar apparatus found in apusomonads and collodictyonids side of B2 and supports an array of superficial microtu- as well. As illustrated in Figure 7, the most recent ancestor bules on the dorsal side of the cell. This typical excavate for all of the major lineages of eukaryotes is inferred to architecture is much more similar to stramenopiles, apuso- have lost some combination of components found in the zoans and collodictyonids than it is to highly diversified typical excavate cytoskeletal configuration. ‘excavate’ lineages like euglenids and parabasalids. Molec- CONCLUSIONS ular phylogenetic data are essentially silent on this issue, but indicate that the Apusozoa and Collodictyonidae are The flagellar apparatus is a complex ultrastructural system only very distantly related to the other eukaryotic super- that is fundamental to the vast majority of eukaryotic cells, groups (Kim et al., 2006; Zhao et al., 2012). This suggests conserved enough to infer homology over large phyloge- that the excavate-like flagellar apparatus is more widely netic distances, and variable enough to distinguish different

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Phycol. 31,96 108. Brown, M.W., Kolisko, M., Silberman, J.D. and Roger, A.J. (2012) Aggrega- elusive, once established, this ancestral flagellar apparatus tive multicellularity evolved independently in the eukaryotic supergroup was independently streamlined several times through the Rhizaria. Curr. Biol. 22, 1123–1127. loss of different traits in different lineages of eukaryotes. Brugerolle, G. (1991) Flagellar and cytoskeletal systems in amitochondrial flagellates: Archamoeba, Metamonada, and Parabasala. Protoplasma, – ACKNOWLEDGEMENTS 164,70 90. Brugerolle, G. (2002a) Cryptophagus subtilis: a new parasite of crypto- This work was supported by grants from the Tula Foundation phytes affiliated with the Perkinsozoa lineage. Eur. J. Protistol. 37, (Centre for Microbial Diversity and Evolution at the University of 379–390. British Columbia) and the Canadian Institute for Advanced Brugerolle, G. 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